WO2024099967A2 - Attenuation of lipopolysaccharide-derived toxicity in a bacterial biomass - Google Patents
Attenuation of lipopolysaccharide-derived toxicity in a bacterial biomass Download PDFInfo
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- WO2024099967A2 WO2024099967A2 PCT/EP2023/080842 EP2023080842W WO2024099967A2 WO 2024099967 A2 WO2024099967 A2 WO 2024099967A2 EP 2023080842 W EP2023080842 W EP 2023080842W WO 2024099967 A2 WO2024099967 A2 WO 2024099967A2
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- Prior art keywords
- biomass
- fraction
- bacterial
- minutes
- biomass fraction
- Prior art date
Links
- 239000002028 Biomass Substances 0.000 title claims abstract description 170
- 230000001580 bacterial effect Effects 0.000 title claims abstract description 89
- 239000002158 endotoxin Substances 0.000 title claims abstract description 42
- 229920006008 lipopolysaccharide Polymers 0.000 title claims abstract description 39
- 231100000419 toxicity Toxicity 0.000 title claims abstract description 35
- 230000001988 toxicity Effects 0.000 title claims abstract description 35
- 238000000034 method Methods 0.000 claims abstract description 59
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- 238000000855 fermentation Methods 0.000 claims description 24
- 241000589346 Methylococcus capsulatus Species 0.000 claims description 22
- 230000004151 fermentation Effects 0.000 claims description 22
- 239000002738 chelating agent Substances 0.000 claims description 17
- 241000894006 Bacteria Species 0.000 claims description 16
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/06—Lysis of microorganisms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/065—Microorganisms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Definitions
- the present invention relates to processes for providing a biomass fraction having reduced toxicity from a bacterial biomass. Further, the present invention relates to a bacterial biomass fraction having a lipopolysaccharide content below 5% w/w of said biomass fraction.
- Methylococcus capsulatus is a non-commensal bacterium found ubiquitously in nature. It metabolizes methane, e.g ., from natural gas, into biomass, CO2 and water. Being rich in protein, M. capsulatus can be used as a protein supplement in animal feed and is also of interest for human consumption. The fermentation of this bacterium as a protein source for both animal and human consumption may contribute to satisfying the world's need for dietary protein in a way which is more environmentally friendly than conventional protein production industries.
- LPS Bacterial lipopolysaccharides
- endotoxins Bacterial lipopolysaccharides
- LPS naturally occurs in bacterial cells and is a structural component thereof; LPS consists of long chains of polysaccharides, which are covalently bound to lipids.
- the bacterial cell membrane constitutes a double lipid layer interspersed with proteins.
- the outer leaflet is LPS, which includes lipid A, a phosphorylated lipid .
- the toxicity of LPS is hypothesised to be mainly due to lipid A, while the polysaccharide part of LPS is generally considered less toxic.
- LPS acts as a pyrogenic compound, generating fever if directly injected into an animal, and 1 to 2 micrograms intravenously injected are lethal to both humans and animals.
- Wassenaar et al. Wangaar TM, Zimmermann K. Eur J Microbiol Immunol (Bp). 2018 Aug 21;8(3) :63-69. doi : 10.1556/1886.2018.00017
- LPS and its toxicity when ingested or injected by animals or humans. Removal of LPS from biomass is described in FI129784.
- LPS lipopolysaccharides
- the resulting product is less harmful, in that it comprises reduced bacterial cell outer membrane component concentrations.
- the present invention relates to a process for providing a biomass fraction having reduced toxicity relative to bacterial biomass, the process comprising the steps of: la. suspending a bacterial biomass in a solution comprising a chelating agent, to provide a first cell suspension, lb. incubating the first cell suspension for a predetermined time, and lc. subjecting the incubated first cell suspension to a separation step to provide a liquid fraction and a first biomass fraction, whereby the first biomass fraction has a reduced toxicity compared to the bacterial biomass.
- the present invention relates to a process for providing a biomass fraction from a bacterial biomass, having reduced toxicity relative to said bacterial biomass, the process comprising the steps of: 2a. lysis of at least a portion of the bacterial cells in the bacterial biomass, so as to provide a second biomass fraction, and 2b. incubating the second biomass fraction for a predetermined time, whereby the second biomass fraction has a reduced toxicity compared to the bacterial biomass.
- the present invention relates to a process for providing a biomass fraction from a bacterial biomass, having reduced toxicity relative to bacterial biomass, combining the process steps of the first and second aspects.
- the process comprising the steps of: 3a. suspending a bacterial biomass in a solution comprising a chelating agent, to provide a first cell suspension, 3b. incubating the first cell suspension for a predetermined time, 3c. subjecting the incubated first cell suspension to a separation step to provide a liquid fraction and a first biomass fraction, followed by 3d.
- steps 3d. and 3e. may take place in any order, so as to provide a third biomass fraction, and 3f. incubating the third biomass fraction for a predetermined time, whereby the third biomass fraction has a reduced toxicity compared to the bacterial biomass.
- divalent cation such as Ca 2+ , Mg 2+ , Cu 2+ , Fe 2+ , Co 2+ , Al 2+
- the present invention relates to a bacterial biomass fraction having a lipopolysaccharide (LPS) content below 5% w/w, more preferably below 2% w/w, more preferably below 1% w/w of said biomass fraction.
- LPS lipopolysaccharide
- the present invention relates to an animal feed comprising or consisting of the bacterial biomass fraction according to the fourth aspect.
- the present invention relates to a human food comprising or consisting of the bacterial biomass fraction according to the fourth aspect.
- Fig. 1 shows an overview of the first process according to the invention.
- Fig. 2 shows an overview of the second process according to the invention.
- Fig. 3 shows an overview of the third process according to the invention, where the process steps 3a-3e are shown with the optional washing and centrifugation steps included.
- biomass refers to a proteinaceous product, which may be in the form of a protein extract and comprises cell wall materials of single celled microorganisms from pure or mixed cultures of algae, yeasts, fungi, or bacteria.
- biomass fraction refers to a fraction, i.e., a part of the biomass.
- single cell protein commonly refers to a proteinaceous product isolated from single celled microorganisms.
- the proteinaceous product may be in the form of a biomass or a protein extract and comprises cell wall materials of single celled microorganisms from pure or mixed cultures of algae, yeasts, fungi, or bacteria.
- the single cell protein is traditionally used as an ingredient or a substitute for protein-rich foods and is suitable for human consumption or as animal feeds.
- Utilizing microorganisms to obtain biomass for use in feed and food results in a product that has a higher proportion of nucleic acids than conventional foods.
- concentration of nucleic acids present in SCP varies depending on the specific microorganism employed, generally about 5 to 18 percent nucleic acids (dry weight) are present in SCP.
- DM refers to "Dry Matter”.
- dry matter and “ash” content is determined according to the A.O.A.C. method (reference A.O.A.C. Standard, 1945).
- dry weight in the context of the dry weight of an M. capsulatus biomass, should be taken to mean the weight of the biomass after all water has been removed from it. This should not be taken to mean that all water has been removed in all embodiments of an M. capsulatus biomass according to the present invention, since water is present in some embodiments. It should rather be understood as a measure that can be used to reproducibly calculate whether a certain biomass falls within the scope of the biomass according to the present invention.
- reduced toxicity refers to a reduced toxicity of the biomass fraction produced by microorganisms, such as pure or mixed cultures of algae, yeasts, fungi, or bacteria, thereby improving the safety of handling and storage of the biomass fractions. This may be e.g., reducing the LPS content in the biomass fraction, thus the reduced toxicity may refer to a reduced LPS content in the biomass fraction compared to an untreated bacterial biomass.
- solution comprising a chelating agent refers to a solution comprising a chelating agent molecule, i.e., a molecule which is able to bond metal ions.
- Chelation typically involves the formation of two or more separate coordinate bonds between a polydentate (multiple bonded) ligand and a single metal ion.
- the invention provides various processes for providing a biomass fraction from a bacterial biomass, having reduced toxicity relative to said bacterial biomass.
- the biomass material (which is typically an aqueous biomass material) is suitably obtained from the fermentation of at least one microorganism, preferably wherein at least one of the microorganisms is a bacterial cell, preferably a methanotroph, more preferably M. capsulatus.
- the microorganism In a fermentation step, the microorganism, or mixture of microorganisms, metabolizes methane into aqueous biomass material, CO2, and water.
- the fermentation occurs in fermentation tanks, and the process is described in detail in, e.g., WO 2017/080987 and WO 2022/008478 hereby incorporated by reference.
- the biomass material is a single-cell protein (SCP) product. It comprises mainly of protein (ca. 60%), and lesser amounts of RIMA and DNA.
- SCP single-cell protein
- the biomass material When isolated from the fermentation step, the biomass material is an aqueous suspension. In this aqueous suspension, the majority of the solid component is cellular material from the microorganism.
- Other components e.g., proteins, nucleic acids, polysaccharides, lipids, LPS or other small molecules
- At least one of the microorganisms used in the fermentation step is suitably a bacterial cell, preferably a Gram negative bacterial cell, preferably a methanotroph, more preferably M. capsulatus. Therefore, the biomass is suitably M. capsulatus biomass.
- Methods capsulatus can mean any strain of bacteria belonging to the M. capsulatus species.
- the strain may be either naturally occurring or developed in a laboratory, such as a genetically modified strain.
- naturally occurring means that the strain has not been genetically modified using genetic engineering techniques. However, it may contain natural modifications or alterations in its genetic material compared to a reference strain, such as alterations that occur randomly during replication.
- the strain is naturally occurring.
- the strain is M. capsulatus (Bath), more preferably the M. capsulatus (Bath) identified under NCIMB 11132. However, it may also be M. capsulatus (Texas) or M. capsulatus (Aberdeen) or a different M. capsulatus strain which is currently known or will be discovered or characterized in the future.
- the methanotrophic bacteria may be provided in a co-fermentation together with one or more heterotrophic bacteria.
- the following heterotrophic bacteria may be particularly useful to co-ferment with M. capsulatus,- Ralstonia sp. ; Bacillus brevis; Brevibacillus agri; Alcaligenes acidovorans; Aneurinibacillus danicus and Bacillus firmus.
- Suitable yeasts may be selected from species of Saccharomyces and/or Candida.
- the preferred heterotrophic bacteria are chosen from Alcaligenes acidovorans (NCIMB 13287), Aneurinibacillus danicus (NCIMB 13288) and Bacillus firmus (NCIMB 13289) and combinations thereof.
- the methanotrophic bacteria and/or the heterotrophic bacteria may be genetically modified.
- the carbon source is converted by the microorganism(s) to biomass material.
- the carbon source comprises methane, and is e.g., natural gas, syngas, or biogas.
- the carbon source is dissolved in the fermentation medium. Fermentation suitably takes place in a U-loop reactor, as described in WO 2010/069313, hereby incorporated by reference.
- a suitable fermentation medium is described in e.g., WO 2018/158322 hereby incorporated by reference.
- the fermentation step has a relatively low Dry Matter content, e.g., below 5%.
- the co-fermentation of M. capsulatus with one or more other organisms may result in a biomass product containing an M. capsulatus biomass as well as a biomass of the one or more other organisms.
- the M. capsulatus is fermented in combination with one or more bacteria selected from : Ralstonia sp., B. brevis, B. agri, A. acidovorans, A. danicus, and B. firmus,- preferably any one or two or all three of: A. acidovorans, A. danicus, and B. firmus; more preferably any one or two or all three of: A. acidovorans (NCIMB 13287), A. danicus (NCIMB 13288), and B. firmus (NCIMB 13289).
- one or more bacteria selected from : Ralstonia sp., B. brevis, B. agri, A. acidovorans, A. danicus, and B. firmus,- preferably any one or two or all three of: A. acidovorans, A. danicus, and B. firmus; more preferably any one or two or all three of: A. acidovorans (NCIMB 13287), A.
- the dry matter content of the biomass material from the fermentation process is between 1- 3%.
- the biomass material may be clarified, and the supernatant from the clarification step may be recycled to the fermenter. Pellets are thus obtained with a dry matter content of 10-18%.
- the fermentation broth in the fermenter may preferably continuously be provided with the required amounts of water and nutrient salts, such as ammonium/ammonia, magnesium, calcium, potassium, iron, copper, zinc, manganese, nickel, cobalt and molybdenum in the form of sulphates, chlorides or nitrates, phosphates and pH controlling components, i.e. acids and/or bases, as normally used by the skilled person, e.g.
- H2SO4 sulphuric acid
- HNO3 nitric acid
- NaOH sodium hydroxide
- KNO3 potassium nitrate
- the biomass material produced from fermentation of natural gas will typically comprise from 60 to 80% by weight crude protein; from 5 to 20% by weight crude fat; from 3 to 12% by weight ash; from 3 to 15% by weight nucleic acids (RNA and DNA).
- RNA and DNA nucleic acids
- the biomass material is subjected to a diafiltration step at this point, to lower the dry matter content to around 6%.
- the first process relates to a process for providing a biomass fraction from a bacterial biomass, having reduced toxicity relative to said bacterial biomass, said process comprising the steps of: la. suspending a bacterial biomass in a solution comprising a chelating agent, to provide a first cell suspension, lb. incubating the first cell suspension for a predetermined time, lc. subjecting the incubated first cell suspension to a separation step to provide a liquid fraction and a first biomass fraction, whereby the first biomass fraction has a reduced toxicity compared to the bacterial biomass.
- Fig. 1 shows the steps of the first process.
- the bacterial biomass is suspended in a solution comprising a chelating agent to provide a first cell suspension.
- the bacterial biomass is derived from fermentation of at least one methanotrophic bacteria, preferably M. capsuiatus.
- the process may further comprise the step of subjecting the bacterial biomass to an initial separation step to remove a first liquid fraction.
- the solution comprising a chelating agent and/or the chelating agent is a metal ion chelating agent, preferably a divalent metal ion chelating agent, preferably EDTA, Ethylenediamine, Glycine, Citric acid, Gluconic acid, Tartaric acid, Hexametaphosphoric acid, Pyrophosphoric acid, Tripolyphosphoric acid, Phytic acid, Free histidine, L-glutamic acid, N,N-diacetic acid, Aspartic acid, citrate, gluconate, or salts thereof, particularly sodium salts thereof, e.g. sodium citrate (TSA), sodium gluconate; preferably sodium EDTA, EDTA, sodium citrate (TSA), sodium gluconate, or mixtures thereof.
- TSA sodium citrate
- TSA sodium gluconate
- TSA sodium gluconate
- TSA sodium gluconate
- TSA sodium gluconate
- TSA sodium gluconate
- TSA
- the first cell suspension is incubated for a predetermined time.
- the incubation of the first cell suspension takes place under one or more of the following conditions: a pH between 5 and 11, preferably between 7 and 9, more preferably between 7.5 and 8.5, a temperature between 5 and 90 °C, preferably between 20 and 90 °C, more preferably between 30 and 90 °C, a time of between 0.5 minutes and 300 minutes, preferably between 1 minute and 180 minutes, preferably between 1 minute and 90 minutes, preferably between 1 minutes and 60 minutes, more preferably between 1 minute and 30 minutes, a concentration of the cells in the first cell suspension of between 10 8 and 10 14 cells/mL, preferably between 10 10 and 10 12 cells/mL.
- the incubated first cell suspension is subjected to a separation step to provide a liquid fraction and a first biomass fraction, whereby the first biomass fraction has a reduced toxicity compared to the bacterial biomass.
- One or more steps of washing and centrifugation of the first biomass fraction may occur after the third step.
- the combined chelation and heating (incubating) steps will maximise/promote the shedding of the outer membrane of the Gram-negative bacteria, therefore shedding of endotoxins while also reducing the nucleic acids in the bacterial biomass product.
- the second process relates to a process for providing a biomass fraction from a bacterial biomass, having reduced toxicity relative to said bacterial biomass, said process comprising the steps of:
- FIG. 2 shows the steps of the first process.
- lysis of at least a portion of the bacterial cells in the bacterial biomass is carried out, to provide a second biomass fraction.
- the lysis step comprises one or more steps selected from heating, cooling, freeze-thawing, sonication, mechanical treatment such as homogenisation, chemical treatment, enzymatic treatment, pressure and filtering, preferably heating.
- the parameters for homogenization are 800 bar and a flow of 80-100 litres/hour. This step is preferably followed by UHT treatment.
- the second biomass fraction is incubated for a predetermined time, whereby the second biomass fraction has a reduced toxicity compared to the bacterial biomass.
- the incubation of the second biomass fraction takes place under one or more of the following conditions: a pH between 5 and 11, preferably between 7 and 9, more preferably between 7.5 and 8.5, a temperature between 5 and 90 °C, preferably between 20 and 60 °C, more preferably between 30 and 50 °C, a time of between 0.5 minutes and 300 minutes, preferably between 1 minute and 180 minutes, preferably between 1 minute and 90 minutes, preferably between 1 minutes and 60 minutes, more preferably between 1 minute and 30 minutes, a concentration of the cells in the first cell suspension of between 10 8 and 10 14 cells/mL, preferably between 10 10 and 10 12 cells/mL.
- the third process relates to a process for providing a biomass fraction from a bacterial biomass, having reduced toxicity relative to said bacterial biomass, in which the steps of the first and second processes are carried out. Therefore, all details of the individual steps described above for the first and second processes are also relevant for the third process described herein.
- the third process thus comprises the steps of:
- steps 3d. and 3e. may take place in any order, to provide a third biomass fraction
- Fig. 3 shows the steps of the third process. Details of the individual steps of suspension, incubation, separation, and other steps in the third process are the same as those specified above for the first and second processes.
- the reduced toxicity may refer to reducing the lipopolysaccharide (LPS) content or potency of the biomass.
- the reduced toxicity may be measured by a conventional cytotoxicity test (such as the limulus amoebocyte lysate (LAL) test), or a quantitative method such as quantification of the intact LPS molecule, or a marker derived from the LPS molecule, by e.g. liquid or gas chromatography followed by mass spectrometry, flame ionisation detection, thermal conductivity detection or another suitable detection method,
- a further advantage is that the resulting product will have reduced outer membrane components and leads to a product with higher protein content.
- the endotoxin is dephosphorylated during the process, thereby their negative effect is attenuated, resulting in a non-toxic product or in a product with reduced toxicity compared to the bacterial biomass.
- bacterial biomass fraction having a lipopolysaccharide (LPS) content below 5% w/w, more preferably below 2% w/w, more preferably below 1% w/w of said biomass fraction.
- LPS lipopolysaccharide
- Such a bacterial biomass fraction can pass the cytotoxicity test due to the reduced/attenuated LPS content present in the bacterial biomass fraction and reduces the chances of adverse effects occurring in the human body during handling or storage of the product.
- An animal feed or human food product may comprise or consist of the bacterial biomass fraction having a lipopolysaccharide (LPS) content below 10% w/w, such as below 7% w/w, more preferably below 5% w/w, more preferably below 2% w/w, more preferably below 1% w/w of said biomass fraction.
- LPS lipopolysaccharide
- Such a product is able to pass the cytotoxicity test. It can be directly fed to animals or provided as a food product (e.g. as ingredient or final product) for humans.
- a bacterial biomass (as obtained by the processes exemplified in e.g. WO 2017/080987 and WO 2022/008478) is subjected to a first centrifugation (using an SPX centrifuge running at 9,000 rpm and discharge time set to 120 seconds, and fed with 500 L per hour and delivering 50-60 L of precipitate per hour and 440-450 L of supernatant per hour) to provide a first bacterial biomass fraction enriched in dry matter (first precipitate, 10-15 % dry matter), and a first supernatant low in dry matter (1-2 % dry matter). This fraction is diluted by 5 fold using water containing 5 mM tetrasodium EDTA.
- the resulting material is heated to 60 °C and immediately subjected to a second centrifugation (using an Alfa Laval CLARA centrifuge running at 9,000 rpm and discharge time set to 120 seconds, and fed with 300 L per hour and delivering 30-40 L of precipitate per hour and 460-470 L of supernatant per hour) to provide a second bacterial biomass fraction (second precipitate, 10- 15 % dry matter) and a second supernatant (1-2 % dry matter).
- second precipitate a content of LPS amounting to 65-70 % relative to the first precipitate on a dry matter basis by quantification of an LPS marker was observed using chromatography.
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Abstract
Processes are described for providing a biomass fraction from a bacterial biomass, having reduced toxicity relative to said bacterial biomass. A bacterial biomass fraction having a lipopolysaccharide (LPS) content below 5% w/w of said biomass fraction is also described. An animal feed comprising or consisting of the bacterial biomass fraction is also provided.
Description
ATTENUATION OF LIPOPOLYSACCHARIDE-DERIVED TOXICITY IN A BACTERIAL BIOMASS
TECHNICAL FIELD
The present invention relates to processes for providing a biomass fraction having reduced toxicity from a bacterial biomass. Further, the present invention relates to a bacterial biomass fraction having a lipopolysaccharide content below 5% w/w of said biomass fraction.
BACKGROUND
The Gram-negative methanotrophic bacterium Methylococcus capsulatus is a non-commensal bacterium found ubiquitously in nature. It metabolizes methane, e.g ., from natural gas, into biomass, CO2 and water. Being rich in protein, M. capsulatus can be used as a protein supplement in animal feed and is also of interest for human consumption. The fermentation of this bacterium as a protein source for both animal and human consumption may contribute to satisfying the world's need for dietary protein in a way which is more environmentally friendly than conventional protein production industries.
Bacterial lipopolysaccharides (LPS, also known as endotoxins) are bacterial toxins which have been described in association with a number of diseases, including liver damage, neurological degeneration, chronic inflammation of the gut and diabetes, among others. LPS naturally occurs in bacterial cells and is a structural component thereof; LPS consists of long chains of polysaccharides, which are covalently bound to lipids. The bacterial cell membrane constitutes a double lipid layer interspersed with proteins. The outer leaflet is LPS, which includes lipid A, a phosphorylated lipid . The toxicity of LPS is hypothesised to be mainly due to lipid A, while the polysaccharide part of LPS is generally considered less toxic.
LPS acts as a pyrogenic compound, generating fever if directly injected into an animal, and 1 to 2 micrograms intravenously injected are lethal to both humans and animals. Wassenaar et al. (Wassenaar TM, Zimmermann K. Eur J Microbiol Immunol (Bp). 2018 Aug 21;8(3) :63-69. doi : 10.1556/1886.2018.00017) describes LPS and its toxicity when ingested or injected by animals or humans. Removal of LPS from biomass is described in FI129784.
As M. capsulatus is a Gram-negative organism, the natural content of lipopolysaccharides (LPS) is high. This poses a potential problem in the manufacturing and processing of bacterial biomass derived from Gram-negative organisms, as inhalation of dust in such processes can lead to LPS entering the bloodstream, which, over a longer period, can result in e.g . liver damage.
It is therefore an object of embodiments of the invention to provide a bacterial biomass with reduced toxicity, as well as processes for the production thereof. Safety in handling and storage of such products can therefore be improved.
SUMMARY
It has been found by the present inventor(s) that by carrying out various process steps on bacterial biomass, the resulting product is less harmful, in that it comprises reduced bacterial cell outer membrane component concentrations.
So, in a first aspect the present invention relates to a process for providing a biomass fraction having reduced toxicity relative to bacterial biomass, the process comprising the steps of: la. suspending a bacterial biomass in a solution comprising a chelating agent, to provide a first cell suspension, lb. incubating the first cell suspension for a predetermined time, and lc. subjecting the incubated first cell suspension to a separation step to provide a liquid fraction and a first biomass fraction, whereby the first biomass fraction has a reduced toxicity compared to the bacterial biomass.
In a second aspect the present invention relates to a process for providing a biomass fraction from a bacterial biomass, having reduced toxicity relative to said bacterial biomass, the process comprising the steps of: 2a. lysis of at least a portion of the bacterial cells in the bacterial biomass, so as to provide a second biomass fraction, and 2b. incubating the second biomass fraction for a predetermined time, whereby the second biomass fraction has a reduced toxicity compared to the bacterial biomass.
In a third aspect the present invention relates to a process for providing a biomass fraction from a bacterial biomass, having reduced toxicity relative to bacterial biomass, combining the process steps of the first and second aspects. In this third aspect, the process comprising the steps of: 3a. suspending a bacterial biomass in a solution comprising a chelating agent, to provide a first cell suspension, 3b. incubating the first cell suspension for a predetermined time, 3c. subjecting the incubated first cell suspension to a separation step to provide a liquid fraction and a first biomass fraction, followed by 3d. adding at least one divalent cation, such as Ca2+, Mg2+, Cu2+, Fe2+, Co2+, Al2+, to the first biomass fraction, 3e. lysis of at least a portion of the bacterial cells in the first biomass fraction, wherein steps 3d. and 3e. may take place in any order, so as to provide a third biomass fraction, and 3f. incubating the third biomass fraction for a predetermined time, whereby the third biomass fraction has a reduced toxicity compared to the bacterial biomass.
In a fourth aspect the present invention relates to a bacterial biomass fraction having a lipopolysaccharide (LPS) content below 5% w/w, more preferably below 2% w/w, more preferably below 1% w/w of said biomass fraction.
In a fifth aspect the present invention relates to an animal feed comprising or consisting of the bacterial biomass fraction according to the fourth aspect.
In a sixth aspect, the present invention relates to a human food comprising or consisting of the bacterial biomass fraction according to the fourth aspect.
LEGENDS TO THE FIGURES
Fig. 1 shows an overview of the first process according to the invention.
Fig. 2 shows an overview of the second process according to the invention.
Fig. 3 shows an overview of the third process according to the invention, where the process steps 3a-3e are shown with the optional washing and centrifugation steps included.
DETAILED DISCLOSURE
Definitions
The term "biomass" refers to a proteinaceous product, which may be in the form of a protein extract and comprises cell wall materials of single celled microorganisms from pure or mixed cultures of algae, yeasts, fungi, or bacteria.
The term "biomass fraction" refers to a fraction, i.e., a part of the biomass.
The term "single cell protein (SCP)" commonly refers to a proteinaceous product isolated from single celled microorganisms. The proteinaceous product may be in the form of a biomass or a protein extract and comprises cell wall materials of single celled microorganisms from pure or mixed cultures of algae, yeasts, fungi, or bacteria. The single cell protein is traditionally used as an ingredient or a substitute for protein-rich foods and is suitable for human consumption or as animal feeds.
Utilizing microorganisms to obtain biomass for use in feed and food results in a product that has a higher proportion of nucleic acids than conventional foods. Although the concentration
of nucleic acids present in SCP varies depending on the specific microorganism employed, generally about 5 to 18 percent nucleic acids (dry weight) are present in SCP.
Throughout this text, the abbreviation "DM" refers to "Dry Matter". In the present context, the terms "dry matter" and "ash" content is determined according to the A.O.A.C. method (reference A.O.A.C. Standard, 1945).
As used herein, the term "dry weight", in the context of the dry weight of an M. capsulatus biomass, should be taken to mean the weight of the biomass after all water has been removed from it. This should not be taken to mean that all water has been removed in all embodiments of an M. capsulatus biomass according to the present invention, since water is present in some embodiments. It should rather be understood as a measure that can be used to reproducibly calculate whether a certain biomass falls within the scope of the biomass according to the present invention.
The term "reduced toxicity" refers to a reduced toxicity of the biomass fraction produced by microorganisms, such as pure or mixed cultures of algae, yeasts, fungi, or bacteria, thereby improving the safety of handling and storage of the biomass fractions. This may be e.g., reducing the LPS content in the biomass fraction, thus the reduced toxicity may refer to a reduced LPS content in the biomass fraction compared to an untreated bacterial biomass.
The term "solution comprising a chelating agent" refers to a solution comprising a chelating agent molecule, i.e., a molecule which is able to bond metal ions. Chelation typically involves the formation of two or more separate coordinate bonds between a polydentate (multiple bonded) ligand and a single metal ion.
As noted, the invention provides various processes for providing a biomass fraction from a bacterial biomass, having reduced toxicity relative to said bacterial biomass.
Fermentation
The biomass material (which is typically an aqueous biomass material) is suitably obtained from the fermentation of at least one microorganism, preferably wherein at least one of the microorganisms is a bacterial cell, preferably a methanotroph, more preferably M. capsulatus.
In a fermentation step, the microorganism, or mixture of microorganisms, metabolizes methane into aqueous biomass material, CO2, and water. The fermentation occurs in fermentation tanks, and the process is described in detail in, e.g., WO 2017/080987 and WO 2022/008478 hereby incorporated by reference.
The biomass material is a single-cell protein (SCP) product. It comprises mainly of protein (ca. 60%), and lesser amounts of RIMA and DNA. When isolated from the fermentation step, the biomass material is an aqueous suspension. In this aqueous suspension, the majority of the solid component is cellular material from the microorganism. Other components (e.g., proteins, nucleic acids, polysaccharides, lipids, LPS or other small molecules) may be dissolved or suspended in the aqueous phase.
At least one of the microorganisms used in the fermentation step is suitably a bacterial cell, preferably a Gram negative bacterial cell, preferably a methanotroph, more preferably M. capsulatus. Therefore, the biomass is suitably M. capsulatus biomass.
The term "Methylococcus capsulatus" or "M. capsulatus" , as used herein, can mean any strain of bacteria belonging to the M. capsulatus species. The strain may be either naturally occurring or developed in a laboratory, such as a genetically modified strain. The term "naturally occurring" means that the strain has not been genetically modified using genetic engineering techniques. However, it may contain natural modifications or alterations in its genetic material compared to a reference strain, such as alterations that occur randomly during replication. Preferably, the strain is naturally occurring. Also preferably, the strain is M. capsulatus (Bath), more preferably the M. capsulatus (Bath) identified under NCIMB 11132. However, it may also be M. capsulatus (Texas) or M. capsulatus (Aberdeen) or a different M. capsulatus strain which is currently known or will be discovered or characterized in the future.
The methanotrophic bacteria may be provided in a co-fermentation together with one or more heterotrophic bacteria. The following heterotrophic bacteria may be particularly useful to co-ferment with M. capsulatus,- Ralstonia sp. ; Bacillus brevis; Brevibacillus agri; Alcaligenes acidovorans; Aneurinibacillus danicus and Bacillus firmus. Suitable yeasts may be selected from species of Saccharomyces and/or Candida. The preferred heterotrophic bacteria are chosen from Alcaligenes acidovorans (NCIMB 13287), Aneurinibacillus danicus (NCIMB 13288) and Bacillus firmus (NCIMB 13289) and combinations thereof. The methanotrophic bacteria and/or the heterotrophic bacteria may be genetically modified.
In the fermentation step, the carbon source is converted by the microorganism(s) to biomass material. Suitably, the carbon source comprises methane, and is e.g., natural gas, syngas, or biogas. During the fermentation step, the carbon source is dissolved in the fermentation medium. Fermentation suitably takes place in a U-loop reactor, as described in WO 2010/069313, hereby incorporated by reference. A suitable fermentation medium is described in e.g., WO 2018/158322 hereby incorporated by reference. The fermentation step has a relatively low Dry Matter content, e.g., below 5%.
The co-fermentation of M. capsulatus with one or more other organisms may result in a biomass product containing an M. capsulatus biomass as well as a biomass of the one or more other organisms.
In some embodiments, the M. capsulatus is fermented in combination with one or more bacteria selected from : Ralstonia sp., B. brevis, B. agri, A. acidovorans, A. danicus, and B. firmus,- preferably any one or two or all three of: A. acidovorans, A. danicus, and B. firmus; more preferably any one or two or all three of: A. acidovorans (NCIMB 13287), A. danicus (NCIMB 13288), and B. firmus (NCIMB 13289).
Further details of the fermentation process are described in WO 2020/245197 and WO 2020/249670, which are hereby incorporated by reference.
The dry matter content of the biomass material from the fermentation process is between 1- 3%. After harvesting the biomass material from the fermentation process, the biomass material may be clarified, and the supernatant from the clarification step may be recycled to the fermenter. Pellets are thus obtained with a dry matter content of 10-18%. The fermentation broth in the fermenter may preferably continuously be provided with the required amounts of water and nutrient salts, such as ammonium/ammonia, magnesium, calcium, potassium, iron, copper, zinc, manganese, nickel, cobalt and molybdenum in the form of sulphates, chlorides or nitrates, phosphates and pH controlling components, i.e. acids and/or bases, as normally used by the skilled person, e.g. sulphuric acid (H2SO4), nitric acid (HNO3), sodium hydroxide (NaOH), potassium nitrate (KNO3). The latter is also a suitable nitrogen source for M. capsulatus. The specific details of the fermentation process, including suitable substrates etc. are described in WO 2000/70014 and WO 2010/069313, which are incorporated by reference.
The biomass material produced from fermentation of natural gas will typically comprise from 60 to 80% by weight crude protein; from 5 to 20% by weight crude fat; from 3 to 12% by weight ash; from 3 to 15% by weight nucleic acids (RNA and DNA).
Optionally, the biomass material is subjected to a diafiltration step at this point, to lower the dry matter content to around 6%.
The first process relates to a process for providing a biomass fraction from a bacterial biomass, having reduced toxicity relative to said bacterial biomass, said process comprising the steps of:
la. suspending a bacterial biomass in a solution comprising a chelating agent, to provide a first cell suspension, lb. incubating the first cell suspension for a predetermined time, lc. subjecting the incubated first cell suspension to a separation step to provide a liquid fraction and a first biomass fraction, whereby the first biomass fraction has a reduced toxicity compared to the bacterial biomass.
Fig. 1 shows the steps of the first process.
Suspension
In a first step of the first process, the bacterial biomass is suspended in a solution comprising a chelating agent to provide a first cell suspension. The bacterial biomass is derived from fermentation of at least one methanotrophic bacteria, preferably M. capsuiatus.
Prior to this step, the process may further comprise the step of subjecting the bacterial biomass to an initial separation step to remove a first liquid fraction.
The solution comprising a chelating agent and/or the chelating agent is a metal ion chelating agent, preferably a divalent metal ion chelating agent, preferably EDTA, Ethylenediamine, Glycine, Citric acid, Gluconic acid, Tartaric acid, Hexametaphosphoric acid, Pyrophosphoric acid, Tripolyphosphoric acid, Phytic acid, Free histidine, L-glutamic acid, N,N-diacetic acid, Aspartic acid, citrate, gluconate, or salts thereof, particularly sodium salts thereof, e.g. sodium citrate (TSA), sodium gluconate; preferably sodium EDTA, EDTA, sodium citrate (TSA), sodium gluconate, or mixtures thereof.
Without being bound by theory, it is thought that metal chelating agents will lead to instability of the outer cell membrane and will reduce nucleic acids in the bacterial biomass. Sequestration of divalent cations is known to destabilise the outer membrane, see e.g. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 243, No. 24, Issue of December 25, pp. 6384- 491, 1968, and FEMS Microbiology Letters 117 (1994) 203-206.
Incubation
In a second step, the first cell suspension is incubated for a predetermined time. The incubation of the first cell suspension takes place under one or more of the following conditions: a pH between 5 and 11, preferably between 7 and 9, more preferably between 7.5 and 8.5, a temperature between 5 and 90 °C, preferably between 20 and 90 °C, more preferably between 30 and 90 °C, a time of between 0.5 minutes and 300 minutes, preferably between 1 minute and 180 minutes, preferably between 1 minute and 90 minutes, preferably between 1 minutes and 60 minutes, more preferably between 1 minute and 30 minutes, a concentration of the cells in the first cell suspension of between 108 and 1014 cells/mL, preferably between 1010 and 1012 cells/mL.
Separation
In a third step, the incubated first cell suspension is subjected to a separation step to provide a liquid fraction and a first biomass fraction, whereby the first biomass fraction has a reduced toxicity compared to the bacterial biomass.
One or more steps of washing and centrifugation of the first biomass fraction may occur after the third step.
The combined chelation and heating (incubating) steps will maximise/promote the shedding of the outer membrane of the Gram-negative bacteria, therefore shedding of endotoxins while also reducing the nucleic acids in the bacterial biomass product.
The second process relates to a process for providing a biomass fraction from a bacterial biomass, having reduced toxicity relative to said bacterial biomass, said process comprising the steps of:
2a. lysis of at least a portion of the bacterial cells in said bacterial biomass, to provide a second biomass fraction,
2b. incubating the second biomass fraction for a predetermined time, whereby the second biomass fraction has a reduced toxicity compared to the bacterial biomass.
Fig. 2 shows the steps of the first process.
Lysis
In a first step of the second process, lysis of at least a portion of the bacterial cells in the bacterial biomass is carried out, to provide a second biomass fraction.
The lysis step comprises one or more steps selected from heating, cooling, freeze-thawing, sonication, mechanical treatment such as homogenisation, chemical treatment, enzymatic treatment, pressure and filtering, preferably heating.
The parameters for homogenization are 800 bar and a flow of 80-100 litres/hour. This step is preferably followed by UHT treatment.
Incubation
In a second step of the second process, the second biomass fraction is incubated for a predetermined time, whereby the second biomass fraction has a reduced toxicity compared to the bacterial biomass.
The incubation of the second biomass fraction takes place under one or more of the following conditions: a pH between 5 and 11, preferably between 7 and 9, more preferably between 7.5 and 8.5, a temperature between 5 and 90 °C, preferably between 20 and 60 °C, more preferably between 30 and 50 °C, a time of between 0.5 minutes and 300 minutes, preferably between 1 minute and 180 minutes, preferably between 1 minute and 90 minutes, preferably between 1 minutes and 60 minutes, more preferably between 1 minute and 30 minutes, a concentration of the cells in the first cell suspension of between 108 and 1014 cells/mL, preferably between 1010 and 1012 cells/mL.
The third process relates to a process for providing a biomass fraction from a bacterial biomass, having reduced toxicity relative to said bacterial biomass, in which the steps of the first and second processes are carried out. Therefore, all details of the individual steps described above for the first and second processes are also relevant for the third process described herein.
The third process thus comprises the steps of:
3a. suspending a bacterial biomass in a solution comprising a chelating agent, to provide a first cell suspension,
3b. incubating the first cell suspension for a predetermined time,
3c. subjecting the incubated first cell suspension to a separation step to provide a liquid fraction and a first biomass fraction, followed by
3d. adding at least one divalent cation, such as Ca2+, Mg2+, Cu2+, Fe2+, Co2+, Al2+, to the first biomass fraction,
3e. lysis of at least a portion of the bacterial cells in the first biomass fraction, wherein steps 3d. and 3e. may take place in any order, to provide a third biomass fraction,
3f. incubating the third biomass fraction for a predetermined time, whereby the third biomass fraction has a reduced toxicity compared to the bacterial biomass.
Fig. 3 shows the steps of the third process. Details of the individual steps of suspension, incubation, separation, and other steps in the third process are the same as those specified above for the first and second processes.
All of the above three processes result in a reduced toxicity compared to the bacterial biomass. The reduced toxicity may refer to reducing the lipopolysaccharide (LPS) content or potency of the biomass. The reduced toxicity may be measured by a conventional cytotoxicity test (such as the limulus amoebocyte lysate (LAL) test), or a quantitative method such as quantification of the intact LPS molecule, or a marker derived from the LPS molecule, by e.g. liquid or gas chromatography followed by mass spectrometry, flame ionisation detection, thermal conductivity detection or another suitable detection method,
As a further, semi-quantitative method, chemical processes can be used to release the fatty acids from LPS, and these can then be subjected to chromatography to separate them and quantify the main fatty acid, as an LPS marker. The fatty acids on LPS are different from the fatty acids on the main lipid population, allowing them to be distinguished from regular fatty
acids. The detoxified product is able to pass the cytotoxicity test due to the reduced/attenuated LPS content.
A further advantage is that the resulting product will have reduced outer membrane components and leads to a product with higher protein content.
Further, by following the process or processes of present invention, the endotoxin is dephosphorylated during the process, thereby their negative effect is attenuated, resulting in a non-toxic product or in a product with reduced toxicity compared to the bacterial biomass.
There is further provided a bacterial biomass fraction having a lipopolysaccharide (LPS) content below 5% w/w, more preferably below 2% w/w, more preferably below 1% w/w of said biomass fraction.
Such a bacterial biomass fraction can pass the cytotoxicity test due to the reduced/attenuated LPS content present in the bacterial biomass fraction and reduces the chances of adverse effects occurring in the human body during handling or storage of the product.
An animal feed or human food product may comprise or consist of the bacterial biomass fraction having a lipopolysaccharide (LPS) content below 10% w/w, such as below 7% w/w, more preferably below 5% w/w, more preferably below 2% w/w, more preferably below 1% w/w of said biomass fraction.
Such a product is able to pass the cytotoxicity test. It can be directly fed to animals or provided as a food product (e.g. as ingredient or final product) for humans.
The present invention has been described with reference to a number of embodiments. The skilled person can combine elements from different embodiments as required. All references cited herein are incorporated by reference.
Examples
In an example embodiment, a bacterial biomass (as obtained by the processes exemplified in e.g. WO 2017/080987 and WO 2022/008478) is subjected to a first centrifugation (using an SPX centrifuge running at 9,000 rpm and discharge time set to 120 seconds, and fed with 500 L per hour and delivering 50-60 L of precipitate per hour and 440-450 L of supernatant per hour) to provide a first bacterial biomass fraction enriched in dry matter (first precipitate,
10-15 % dry matter), and a first supernatant low in dry matter (1-2 % dry matter). This fraction is diluted by 5 fold using water containing 5 mM tetrasodium EDTA. The resulting material is heated to 60 °C and immediately subjected to a second centrifugation (using an Alfa Laval CLARA centrifuge running at 9,000 rpm and discharge time set to 120 seconds, and fed with 300 L per hour and delivering 30-40 L of precipitate per hour and 460-470 L of supernatant per hour) to provide a second bacterial biomass fraction (second precipitate, 10- 15 % dry matter) and a second supernatant (1-2 % dry matter). In the second precipitate, a content of LPS amounting to 65-70 % relative to the first precipitate on a dry matter basis by quantification of an LPS marker was observed using chromatography.
Claims
1. A process for providing a biomass fraction from a bacterial biomass, having reduced toxicity relative to said bacterial biomass, said process comprising the steps of: la. suspending a bacterial biomass in a solution comprising chelating agent, to provide a first cell suspension, lb. incubating the first cell suspension for a predetermined time, lc. subjecting the incubated first cell suspension to a separation step to provide a liquid fraction and a first biomass fraction, whereby the first biomass fraction has a reduced toxicity compared to the bacterial biomass.
2. A process for providing a biomass fraction from a bacterial biomass, having reduced toxicity relative to said bacterial biomass, said process comprising the steps of:
2a. lysis of at least a portion of the bacterial cells in said bacterial biomass, to provide a second biomass fraction,
2b. incubating the second biomass fraction for a predetermined time, whereby the second biomass fraction has a reduced toxicity compared to the bacterial biomass.
3. A process for providing a biomass fraction from a bacterial biomass, having reduced toxicity relative to said bacterial biomass, said process comprising the steps of:
3a. suspending a bacterial biomass in a solution comprising a chelating agent, to provide a first cell suspension,
3b. incubating the first cell suspension for a predetermined time,
3c. subjecting the incubated first cell suspension to a separation step to provide a liquid fraction and a first biomass fraction, followed by
3d. adding at least one divalent cation, such as Ca2+, Mg2+, Cu2+, Fe2+, Co2+, Al2+, to the first biomass fraction,
3e. lysis of at least a portion of the bacterial cells in said first biomass fraction, wherein steps 3d. and 3e. may take place in any order, so as to provide a third biomass fraction,
3f. incubating the third biomass fraction for a predetermined time, whereby the third biomass fraction has a reduced toxicity compared to the bacterial biomass.
4. The process according to any one of claims 1 or 3, further comprising the step of subjecting said bacterial biomass to an initial separation step to remove a first liquid fraction prior to step la or 3a.
5. The process according to any one of claims 1 or 3, further comprising one or more steps of washing and centrifugation of the first biomass fraction, after step lc or step 3c, and before any subsequent steps 3d-3f.
6. The process according to any one of claims 3-5, wherein at least one divalent cation is added to the first biomass fraction in the form of an aqueous solution of said divalent cation(s).
7. The process according to any one of claims 1-6, wherein incubation of the first cell suspension in step lb or step 3b takes place under one or more of the following conditions: a pH between 5 and 11, preferably between 7 and 9, more preferably between 7.5 and 8.5, a temperature between 5 and 90 °C, preferably between 20 and 90 °C, more preferably between 30 and 90 °C, a time of between 0.5 minutes and 300 minutes, preferably between 1 minute and 180 minutes, preferably between 1 minute and 90 minutes, preferably between 1 minutes and 60 minutes, more preferably between 1 minute and 30 minutes, a concentration of the cells in the first cell suspension of between 108 and 1014 cells/mL, preferably between 1010 and 1012 cells/mL.
8. The process according to any one of claims 3-7, wherein or incubating the second biomass fraction in step 2b or the third biomass fraction in step 3f takes place under one or more of the following conditions; a pH between 5 and 11, preferably between 7 and 9, more preferably between 7.5 and 8.5, a temperature between 5 and 90 °C, preferably between 20 and 60 °C, more preferably between 30 and 50 °C, a time of between 0.5 minutes and 300 minutes, preferably between 1 minute and 180 minutes, preferably between 1 minute and 90 minutes, preferably between 1 minutes and 60 minutes, more preferably between 1 minute and 30 minutes, a concentration of the cells in the first cell suspension of between 108 and 1014 cells/mL, preferably between 1010 and 1012 cells/mL.
9. The process according to any one of claims 2-8, wherein the lysis step (2a; 3e) comprises one or more steps selected from heating, cooling, freeze-thawing, sonication, mechanical treatment such as homogenisation, chemical treatment, enzymatic treatment, pressure and filtering, preferably heating.
10. The process according to any one of claims 1, 3-9, wherein the chelating agent is a metal ion chelating agent, preferably a divalent metal ion chelating agent, preferably EDTA, Ethylenediamine, Glycine, Citric acid, Gluconic acid, Tartaric acid, Hexametaphosphoric acid, Pyrophosphoric acid, Tripolyphosphoric acid, Phytic acid, Free histidine, L-glutamic acid, N,N- diacetic acid, Aspartic acid, citrate, gluconate, or salts thereof, particularly sodium salts thereof, e.g. sodium citrate (TSA), sodium gluconate; preferably sodium EDTA, EDTA, sodium citrate (TSA), sodium gluconate, or mixtures thereof.
11. A bacterial biomass fraction having a lipopolysaccharide (LPS) content below 5% w/w, more preferably below 2% w/w, more preferably below 1% w/w, more preferably below 0.5% w/w, more preferably below 0.2% w/w, more preferably below 0.1% w/w of said biomass fraction.
12. The process according to any one of claims 1-10, or the bacterial biomass according to claim 11, wherein the bacterial biomass is derived from fermentation of at least one methanotrophic bacteria, preferably Methylococcus capsulatus.
13. An animal feed comprising or consisting of the bacterial biomass fraction according to claim 12.
14. A human food product comprising or consisting of the bacterial biomass fraction according to claim 12.
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| EP22386074 | 2022-11-07 |
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| WO2000070014A1 (en) | 1999-05-18 | 2000-11-23 | Ebbe Busch Larsen | U-shape and/or nozzle-u-loop fermentor and method of carrying out a fermentation process |
| WO2010069313A2 (en) | 2008-12-15 | 2010-06-24 | Ebbe Busch Larsen | U-shape and/or nozzle u-loop fermenter and method of fermentation |
| WO2017080987A2 (en) | 2015-11-09 | 2017-05-18 | Unibio A/S | Process for improved fermentation of a microorganism |
| WO2018158322A1 (en) | 2017-03-01 | 2018-09-07 | Unibio A/S | New fermentation medium for growth of methanotrophic bacteria and method for producing said medium |
| WO2020245197A1 (en) | 2019-06-07 | 2020-12-10 | Unibio A/S | Method for optimizing a fermentation process |
| WO2020249670A1 (en) | 2019-06-13 | 2020-12-17 | Unibio A/S | Method for controlling a fermentation process |
| WO2022008478A2 (en) | 2020-07-07 | 2022-01-13 | Unibio A/S | Process for producing single cell protein |
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| WO2000070014A1 (en) | 1999-05-18 | 2000-11-23 | Ebbe Busch Larsen | U-shape and/or nozzle-u-loop fermentor and method of carrying out a fermentation process |
| WO2010069313A2 (en) | 2008-12-15 | 2010-06-24 | Ebbe Busch Larsen | U-shape and/or nozzle u-loop fermenter and method of fermentation |
| WO2017080987A2 (en) | 2015-11-09 | 2017-05-18 | Unibio A/S | Process for improved fermentation of a microorganism |
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